GSM7925237: Lung-tdT+, bleo injuryed 2 months, scRNAseq; Mus musculus; RNA-Seq
The lung epithelial cells isolation procedure included two rounds of digestion by two different enzyme cocktail (enzyme mixture 1: collagenase type I (500 U ml-1, Gibco, 17100–017), elastase (4 U ml-1, Worthington Biochemical Corporation, LS002279), and dispase (5 U ml-1, BD Biosciences, 354235) in DMEM/F12 (Gibco, 10565018); enzyme mixture 2: 0.1% Trypsin-EDTA (Gibco, 25200072) and DNase I (0.33 U ml-1; Worthington Biochemical Corporation LS002139) in DMEM/F12 (Gibco, 10565018)). In brief, the mice was euthanized by CO2 inhalation and then 10 ml of cold PBS was perfused through the right ventricle. After injecting 1.5 ml of enzyme mixture 1 through trachea, the lung lobes was collected and cut in to pieces, and additional 2.5 ml of enzyme mixture 1 was added before incubating the sample at 37 °C for 20 minutes on orbital shaker (~100 r.p.m). The digestion was ended by adding equal volume of cold DMEM/F12 with 10% of FBS (Gibco, 10099141) to the digestion medium, and the tissue was disturbed by pipetting before centrifuged at 1000g and 4 °C for 5 minutes. The supernatant was discarded and the pellet was resuspended with 2 ml of enzyme mixture 2 for incubating at 37 °C for 20 minutes. The digestion was stopped by adding equal volume of DMEM/F12 containing of 10% FBS and then filtered with 100 μm strainer and centrifuged at 1000g and 4 °C for 5 minutes. The pellet was resuspended by 2-3 ml of red blood cell lysis buffer (eBioscience, 00-4333-57) and incubated at room temperature for 3 minutes and then was stopped by adding 3 ml of cold PBS. The sample was filtered through 40 μm stainer and then centrifuged and resuspended in PBS. For MACS and FACS combined isolation, the cells was incubated with Epcam-APC (1:100, eBioscience, 17-5791-82) at 4 °C for 30 minutes and ended by adding cold PBS and centrifuged at 1000g and 4 °C for 5 minutes. For each adult lung, the pellet was resuspended in 80 μl of PBS and followed by 320 μl of anti-APC MicroBeads (Miltenyi Biotec, 130-090-855), then mixed the cells and beads well by pipetting and incubated at 4 °C for 15 minutes. After washing the sample with PBS and centrifuging at 1000g and 4 °C for 5 minutes, the pellet was resuspended in 500 ul of PBS and performed MACS positive isolation by autoMACS Pro Separator (Miltenyi Biotec). The positive selected cells were stained with DAPI and then performed FACS isolation by gating DAPI– Epcam+ tdTomato+ cells. The cell suspense was loaded in 10x Chromium controller to generate GEMs, which were further processed into single cell 5ʹ gene expression libraries using Chromium Single Cell 5ʹ Library Kit (v2 Chemistry).